Protein phosphorylation and dephosphorylation constitute a fundamental mechanism used by cells to control biological processes; it has been estimated that a third of all cellular proteins are phosphorylated. The biological importance and ubiquity of phosphoryl transfer has motivated considerable effort toward understanding the mechanisms of the enzymes that catalyze these reactions. The ubiquity of phosphatases in biological pathways has arisen in spite of the fact that the uncatalyzed hydrolysis of a phosphate ester is among the slowest reactions known. This project will explore several potential factors that may contribute to the extremely high catalytic efficiencies of protein-tyrosine phosphatases (PTPases). The potential for local environmental effects to destabilize the ionic phosphate ester substrates, and thus to contribute to enzymatic catalysis, will be evaluated using [18]O isotopic shifts in [31]P NMR to study desolvation and counterion effects in solution, and by measurement of equilibrium isotope effects on binding of substrates to catalytically disabled phosphatases. Separately, the role of a protein conformational change that is a part of the catalytic mechanism common to PTPases will be studied, using several kinetic and thermodynamic methods. The hypothesis that protein conformational isomerization may be coupled to chemical catalysis, and thus is a source of catalytic power, will be tested. Finally, a mechanistic study of a PTPase from a thermophilic organism will be carried out. Phosphate monoester hydrolysis shows an unusually high temperature dependence (the rate of uncatalyzed alkyl phosphate hydrolysis increases approximately 30-fold for each 15 degrees C). Furthermore, the entropy of activation is positive (favorable) in contrast to the situation with other biologically relevant hydrolytic reactions. Thus, overcoming entropy cannot be a significant source of catalytic efficiency for phosphatases. These factors make the enzymology of phosphatases from thermophilic organisms interesting in their own right, as well as a vehicle to study the thermodynamic contributions to catalysis, which may differ in high-temperature and low-temperature organisms.